This invention relates to a method of demarcating two particle groups the boundary of which is not perfectly clear physiologically or morphologically, as is the case with mature red blood cells and reticulocytes which correspond to immature blood cells, on a plot of a one-dimensional distribution such as a particle distribution.
Immature red blood cells in a blood are referred to as reticulocytes and ordinarily make up about 0.7-2.2% of the total number of red blood cells. Measuring reticulocyte count is useful in substantiating diagnoses of acute internal bleeding, hemolytic anemia, aplastic anemia and another illnesses, in monitoring progress following the administration of medication, etc., and is thus of great importance in the field of medical examination.
In order to count reticulocytes, the conventional approach is to use a method in which a blood smear dyed with a basic dye such as new methyl blue or brilliant cresyl blue is observed and the percentage of the colored reticulocytes contained in the total number of red blood cells is determined by visual computation.
This method requires a great amount of time and labor for such pre-treatment as dying of the blood sample and for visual computation following dying and therefore is unsuitable where a large number of samples is concerned.
Accordingly, several methods of automating the counting of reticulocytes by utilizing flow cytometry have been proposed. For example, the specification of U.S. Pat. No. 4,325,706 describes a method of measuring a blood sample, which is fluorescently dyed with acridene orange, by means of a flow cytometer, and determining the reticulocyte count based on a plot of a two-dimensional distribution prepared from signals indicative of fluorescence and scattering detected from individual particles. FIG. 8 is a view of FIG. 8B of the drawings in the above-mentioned specification. The horizontal axis represents the signal strength of red fluorescence detected from the particles, and the vertical axis represents the signal strength of scattered light detected from the particles. A red blood cell (RBC) particle group and a reticulocyte (RETICS) particle group in the center of the view are demarcated from a platelet (PLT) particle group by a diagonally extending linear discriminant. Next, the platelets demarcated by the discriminant are removed, after which a one-dimensional distribution having the red fluorescense signal strength plotted along the horizontal axis is drawn regarding the remaining red blood cell particles and reticulocyte particles. The result is the one-dimensional distribution shown in FIG. 9 (which is taken from FIG. 9 of the above-mentioned specification).
In the aforesaid U.S. patent, a normal distribution curve is estimated from the curve on the right side of the red blood cell (RBC) region in FIG. 9, and the curve on the right side of the red blood cell distribution is extrapolated (the curve indicated by the dashed line in FIG. 9). This is based on an assumption that a frequency distribution of the red blood cells conforms to a normal distribution. A point at which this extrapolated curve departs from the original frequency distribution curve is determined as a threshold value, a region having a fluorescence intensity above this threshold value and bounded by the original frequency distribution curve and the above-mentioned extrapolated curve serves as a reticulocyte (RETIC) region, the number of particles contained in this region is counted, and this number is adopted as the reticulocyte count.
In a case where the one-dimensional frequency distribution is formed by partially superimposing a plurality of particle groups having completely different shapes, properties and origins, it is possible to estimate the distribution of each particle group and count the number of particles in each particle group. For example, the specification of Japanese Patent Application Laid-Open (KOKAI) No. 63-32347 describes, as one example, analyzing a red blood cell particle size distribution (a one-dimensional frequency distribution) of blood in which blood cells of two different sizes are mixed owing to a blood transfusion, estimating the particle size distribution of each of these two particle groups, and determining the red blood cell count in each group. FIG. 10 illustrates the particle size distribution for this case, in which the dashed lines represent the estimated distribution.
However, the relationship between red blood cells and reticulocytes within the same specimen is not a relationship between two entirely different particle groups. That is, in the course of red blood cell maturation, a reticulocyte is an immature red blood cell in a stage just prior to becoming a mature red blood cell. (In the present specification, the term "red blood cell" shall refer to a mature red blood cell throughout.) A reticulocyte corresponding to an immature red blood cell usually loses ribonucleic acid (RNA) in one to two days to become a mature red blood cell. The maturation of reticulocytes into mature red blood cells is a continuous process and several phases of reticulocytes, ranging from those that are closer to mature red blood cells than others to those that are more immature than others, exist at any one time. With reference to FIG. 9, those reticulocytes in the reticulocyte particle group that emit stronger fluorescence exhibit greater immaturity, the intensity of fluorescence weakening as the reticulocytes mature. By the time the intensity of fluorescence approaches that for mature red blood cells, the region entered is one in which reticulocytes cannot be distinguished from mature red blood cells. Accordingly, it is fair to say that the one-dimensional distribution of FIG. 9 represents a distribution of continuously maturing cells rather than a superposition of distributions of two particle groups having completely different origins. In cases such as this, it is difficult in actual practice to make an estimate about two clearly different particle groups as shown in FIG. 10 and the concept is unreasonable. Though the extrapolated curve in which the red blood cell distribution is estimated by a normal distribution is drawn in FIG. 9, the boundary between the red blood cell distribution and the reticulocytes would be an obscure one, not the distinct boundary shown.